Primary Hyperoxaluria (PH) is a rare, genetic disorder that is characterized by an increased urinary oxalate excretion, the formation of calcium oxalate kidney stones, and in severe cases renal failure. In the most extreme cases, some develop nephrocalcinosis and renal failure as infants with a poor survival outlook. Our research suggests that hydroxyproline metabolism makes a major contribution to the increased oxalate synthesis that occurs in PH. This metabolism occurs in the mitochondrion and is aberrant in a recently identified form of the disease, Type 3, where the activity of the enzyme, 4-hydroxy-2-oxoglutarate aldolase (HOGA), a component of the degradation pathway, is deficient. A deficiency in another mitochondrial enzyme, glyoxylate reductase, is associated with Type 2 disease. In Type 1 disease, glycolate-glyoxylate cycling occurs in the liver when glycolate produced in mitochondria from hydroxyproline metabolism is oxidized in peroxisomes to glyoxylate and reduced back to glycolate in the cytoplasm because of the absence of AGT. We hypothesize that the hydrogen peroxide produced with this cycling contributes to mitochondrial dysfunction due to an increased generation of reactive oxygen species. We further hypothesize that the altered metabolism in these types of PH may result in an altered concentration of oxalate, glyoxylate and glycolate in mitochondria and the cytosol. Changes in ion levels, particularly oxalate and calcium, could further modify mitochondrial properties. In this proposal, we will use genetically modified mice to determine how the changes in enzyme composition associated with PH alter mitochondrial properties in hepatocytes and renal proximal tubule cells (RPTC). The first specific aim will determine the phenotype of Hoga1 knock-out mice and examine how the substrate, HOG, is split when HOGA is absent. We hypothesize that an alternative aldolase is able to split HOG when its concentration increases sufficiently. The second specific aim will examine mitochondrial properties and metabolic changes that occur in intact hepatocytes and RTPC from normal and genetically modified mice using an XF-analyzer. Mitochondrial quality will also be assessed in liver and kidney tissue of the mouse models and in liver tissue from PH patients receiving a transplant. Whether a mitochondrial specific drug such as MitoQ can offset any adverse changes will be investigated. The third specific aim will identify changes that occur in mitochondria isolated from these mice when they metabolize hydroxyproline and glyoxylate. These experiments will illuminate the metabolism associated with the increased oxalate synthesis that occurs in PH, highlight the role played by mitochondria in the disease process, and illustrate important differences between liver and kidney mitochondria. This research should lead to novel approaches to decrease excessive oxalate synthesis and modify mitochondrial dysfunction in PH.